Annexin proteins and autoantibodies as serum markers for cancer

ABSTRACT

The present invention relates to screening methods for diagnosis, prognosis, or susceptibility to cancer in a subject by means of detecting the presence of serum autoantibodies to specific annexin protein antigens in sera from subjects. The present invention also provides screening methods for diagnosis and prognosis of cancer in a subject by means of detecting increased expression levels of annexin proteins in biological samples of the subject. The method of the invention can also be used to identify subjects at risk for developing cancer. The method of the invention involves the use of subject derived biological samples to determine the occurrence and level of expression of annexin proteins or expression of annexin derived peptides or antigens, and/or the occurrence and level of circulating autoantibodies to specific annexin protein antigens. The present invention further provides for kits for carrying out the above described screening methods. Such kits can be used to screen subjects for increased levels of annexin proteins, or for the detection of autoantibodies to annexin proteins, as a diagnostic, predictive or prognostic indicator of cancer.

1. INTRODUCTION

The present invention relates to screening methods for diagnosis,prognosis, or susceptibility to cancer in a subject by means ofdetecting the presence of serum autoantibodies to specific annexinprotein antigents in sera from subject. The present invention alsoprovides screening methods for diagnosis and prognosis of cancer insubject by means of detecting increased expression levels of annexinproteins in biological samples of the subject. The method of theinvention can also be used to identify subject at risk for developingcancer. The method of the invention involves the use of subject derivedbiological samples to determine the ocurrence and level of expression ofannexin proteins or expression of annexin derived peptides of antigent.and/or the occurrence and level of circulating autoantibodies tospecific annexin protein antigens. The present invention furtherprovides for kits for carrying out the above described screeningmethods. Such kits can be used to screen subjects for increased levelsof annexin proteins, or for the detection of autoantibodies to annexinproteins, as a diagnostic, predictive or prognostic indicator of cancer.The invention is demonstrated by way of examples in which elevatedlevels of annexin proteins, and elevated levels of circulatingautoantibodies reactive against annexin proteins, have been observed inthe sera of cancer subjects.

2. BACKGROUND OF THE INVENTION

A number of cellular proteins have been demonstrated to occur atincreased levels in body fluids of subjects with different types ofcancer. The increased levels of such proteins in cancer subjects providediagnostic and prognostic assays for the presence of cancer. Forexample, elevated serum levels of prostate specific antigen (PSA) isfrequently used as an indicator of the presence of prostate cancer inmen.

Autoantibodies to normal or modified cellular proteins are known to beproduced by patients in certain diseases such as autoimmune diseases andcardiovascular-related disorders, in some cases even before the diseasehas produced overt symptoms. There is also increasing evidence for ahumoral immune response to cancer in humans, as demonstrated by theidentification of antibodies against a number of intracellular andsurface antigens in patients with various tumors (Gourevitch et al.,1995, Br. J. Cancer 72:934-938; Yamamoto et al., 1996, Int. J. Cancer,69:283-289; Stockert et al., 1998, J. Exp. Med. 187:1349-1354; Gure etal., 1998, Cancer Res. 58:1034-1041). For example, somatic alterationsin the p53 gene elicit a humoral response in 30-40% affected patients(Soussi, 1996, Immunol. Today 17:354-356). In some instances, thedetection of anti-p53 antibodies can predate the diagnosis of cancer(Lubin et al., 1995, Nat. Med. 7:701-702; Cawley et al., 1998,Gastroenterology 115:19-27). U.S. Pat. No. 5,405,749 discloses a methodof screening for cancer-associated retinopathy autoantigen and testing apatient's serum for autoantibody to the autoantigen. In addition,increases in relative rates of synthesis of major cytoskeletal proteinshave been observed on the surface of leukemic cells and of lymphocytestransformed by mitogens and Epstein-Barr Virus (Bachvaroff, R. J. etal., 1980, Proc. Natl Acad. Sci. 77: 4979-4983).

The majority of tumor derived antigens that have been identified andthat elicit a humoral response are not the products of mutated genes.They include differentiation antigens and other gene products that areoverexpressed in tumors (Old and Chen, 1998, J. Exp. Med. 187:1163-1167). It is not clear why only a subset of patients with a tumortype develop a humoral response to a particular antigen. Factors thatinfluence the immune response may include variability among individualsin major histocompatibility complex molecules. It is also possible thatproteins may become immunogenic after undergoing a post-translationalmodification, a process which may be variable among tumors of a similartype.

Lung cancer is the most common cancer in the United States and accountsfor over one fourth (28%) of cancer deaths in the US (Travis et al.,1996, Cancer 77:2464-2470). A number of molecular alterations includingc-myc amplification, Ki-ras or p53 mutations have been identified thatmay affect tumor behavior (Mao et al., 1994, Cancer Res 54:1634-1637,Mills et al., 1995, J. Natl. Cancer Inst. 87:1056-1060, Gao et al.,1997, Carcinogenesis 18:473-478). Serum autoantibodies against theproduct oncogenes and tumor suppressor genes, such as c-myc (Ben-Mahrezet al., 1990, Int. J. Cancer 46:35-38), c-myb (Sorokine et al., 1991,Int. J. Cancer 47:665-669), c-erbB-2 (Pupa et al., 1993, Cancer Res53:5864-5866), ras (Takahashi et al., 1995, Clin. Cancer 1:107) and p53(Peyrat et al., 1995, Lancet 345:621-622; Iizasa et al., 1998, CancerImmunol. Immunother. 46:345-349), have been reported in patients withvarious malignant diseases. Autoantibodies against L-myc oncogeneproducts have been reported in 10% of sera from patients with lungcancer (Yamamoto et al., 1996, Int. J. Cancer 69:283-289). Serumautoantibodies against p53 have also been detected in sera ofnon-small-cell lung cancer patients (NSCLC) (Iizasa et al., 1998, CancerImmunol. Immunother. 46:345-349). Elevated serum titers of anti-p53autoantibodies were present in approximately 20% of the cases of(NSCLC), and the occurrence of these autoantibodies reflect the presenceof p53 mutations and p53 over expression (Yamamoto et al., 1996, Int. J.Cancer 69:283-289).

The detection of autoantibodies to cellular antigens and theidentification of proteins that have elicited autoantibodies has beenaccomplished using a variety of approaches. For example, ProliferatingCell Nuclear Antigen (PCNA) was first described as a nuclear antigenwhich bound antibodies from some patients with lupus erythematosus(Miyachi, K., Fritzler, M. J., and Tan, E. M., 1978, J. Immunol121:2228-2234). It was subsequently observed that resting lymphocytesdid not react with the antibody, in contrast to mitogen stimulatedlymphocytes which displayed nuclear staining. This ultimately led to theidentification of the protein, designated PCNA which is recognized bythis autoantibody in lupus (Tan, E. M., Ogata, K., and Takasaki, Y.,1987, J. Rheumatol., 13:89-96). In some other cases, candidate proteinsare singled out and investigated with respect to their ability to induceantibodies in patients, as was investigated for p53 (Crawford, L. V.,Firm, D. C., Bulbrook, R. D., 1984, Int J Cancer 30:403-408). Inaddition, a technique called SEREX relies on serological analysis ofrecombinant cDNA expression libraries to identify tumor antigens (Old,L., et al. 1998, J. Exp. Med. 187:1163-1167). Thus, many approaches havebeen followed to search for proteins against which autoantibodies may beproduced.

Annexins are a family of calcium-dependent phospholipid-binding proteinsthat are expressed ubiquitously in different tissues and cell types ofhigher and lower eukaryotes (Benz, J. and Hofmann, A., 1997, Biol. Chem378:177-183). At least twelve annexin proteins have been identified.Among the many roles suggested for the annexin family of proteins, thoseimplicating the proteins in regulated exocytosis remain the mostconvincing (Donnelly, S R and Moss S E, Cell., 1997, Mol. Life Sci.53:533-538). A typical annexin protein is characterized by two distinctfeatures, (i) Ca²⁺-dependent binding to phospholipids; and (ii) thepresence of a conserved sequence element of about 70 amino acids whichis repeated four or eight times in a given member of the family.Immunocytochemical studies of annexins have shown that they residesubadjacent to plasma membranes, near calcium-sequestering intracellularorganelles (Gerke, V and Moss, S E, 1997, Biochimica et Biophysica Acta1357:129-154). Physical properties associated with annexins includeinhibition of phospholipase A₂, anticoagulant activity, binding tocytoskeletal proteins, aggregation of membranes and vesicles andcalcium-selective channel activity. Increased levels of annexin havebeen found to be associated with a number of diseases including multiplesclerosis and experimental neuritis.

3. SUMMARY OF THE INVENTION

It is an object of the present invention to provide screening methodsfor the diagnostic and prognostic evaluation of cancer, for theidentification of subjects possessing a predisposition to cancer, andfor monitoring patients undergoing treatment of cancer, based on thedetection of elevated levels of annexin autoantibodies in biologicalsamples of subjects. The invention also provides methods for detectingoverproduction of annexin proteins and/or overproduction of groups ofannexin proteins as a diagnostic or prognostic indicator of cancer.

The present invention relates to diagnostic evaluation and prognosis ofcancer by detecting autoantibodies to annexin protein antigens in theserum of subjects with cancer or with precancerous lesions. Thedetection of increased serum levels of autoantibodies to annexinproteins constitutes a novel strategy for screening, diagnosis andprognosis of cancer.

The present invention provides for the use of the annexin proteinantigens in immunoassays designed to detect the presence of serumautoantibodies to the annexin protein antigens. Such immunoassays can beutilized for diagnosis and prognosis of cancer. In accordance with theinvention, measurement of annexin autoantibody levels in a subject'sserum can be used for the early diagnosis of cancer. Moreover, themonitoring of serum autoantibody levels can be used prognostically tostage progression of the disease.

The invention further relates to assays developed to detect the level ofannexin proteins in a subject's sample. Such assays include immunoassayswherein the annexin proteins are detected by their interaction withanti-annexin specific antibodies. For example, annexin antibodies orfragments of antibodies may be used to quantitatively detect thepresence and amount of annexin proteins in a subject's sample.

The invention also relates to the use of annexin proteins as antigens toimmunize patients suffering from diseases characterized by increasedexpression levels of the annexin protein antigens. Stimulation of animmunological response to such antigens, is intended to elicit a moreeffective attack on tumor cells; such as inter alia inhibiting tumorcell growth or facilitating the killing of tumor cells. Theidentification of autoantibodies to annexin protein antigens associatedwith particular cancers provides a basis for immunotherapy of thedisease.

The invention further provides for pre-packaged diagnostic kits whichcan be conveniently used in clinical settings to diagnose patientshaving cancer or a predisposition to developing cancer. The kits canalso be utilized to monitor the efficiency of agents used for treatmentof cancer. In one embodiment of the invention, the kit comprisescomponents for detecting and/or measuring the levels of autoantibodiesdirected toward annexin antigens in a sample. In a second embodiment,the kit of the invention comprises components which detect and/ormeasure annexin antigens in the biological sample.

The present invention is based on the discovery that expression levelsof Annexin I and II are increased in tumor tissue samples derived fromsubjects with pulmonary adenocarcinoma or squamous cell lung cancer.Additionally, increased levels of autoantibodies against Annexin I andII were detected in the serum of the subjects with lung adenocarcinomaand squamous cell carcinoma. The finding that levels of annexin proteinsand annexin autoantibodies are increased in samples derived from cancersubjects provides a basis for development of diagnostic and prognosticmethods as well as a means for monitoring the efficacy of varioustherapeutic treatments for cancer.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Two-dimensional gel electrophoresis of A549 cell lysates. Thegel was stained with silver to visualize the proteins.

FIG. 2. Western blot of a two-dimensional gel separation of a A549 celllysate treated with serum from a lung cancer patient. Reactivity wasobserved in a group of contiguous protein spots designated A1 with a pIof between 6.3 and 6.8 and a molecular weight of 37 kDa.

FIG. 3. IgM immunoreactivity against lung cancer proteins. Western blotof a two-dimensional gel separation of A549 lysates treated with serumfrom lung cancer patients. Membranes were subsequently incubated with ahorseradish peroxidase-conjugated sheep anti-human IgM antibody.

FIG. 4. IgG subtype immunoreactivity against lung cancer patient sera.Western blot of a two-dimensional gel separation of A549 lysates withserum from lung cancer patients. Membranes were subsequently hybridizedwith horseradish peroxidase-conjugated mouse anti-human IgG1, IgG2, IgG3or IgG4 antibody.

FIG. 5. Specificity of A1 and A2 IgG antibodies to lung cancer patientsera. Membranes containing A549 lysates were hybridized with sera frompatients with melanoma, breast cancer, liver cancer or esophagal cancer.None of the sera exhibited IgG based immunoreactivity against A2. A1proteins were observed with sera from patients with esophagal cancer(5/8), breast cancer (1/11), but were absent with sera from patientswith melanoma, (0/7) and liver cancer (0/11).

FIG. 6. Anti-Annexin I, and anti-Annexin II antibodies areimmunoreactive against proteins in the A1 and A2 spots respectively.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention achieves a highly desirable objective, namelyproviding methods for the diagnostic and prognostic evaluation ofsubjects with cancer and the identification of subjects exhibiting apredisposition to developing cancer. The assays of the inventioncomprise methods designed to detect increased levels of annexin proteinproduction, or the presence of annexin autoantibodies, in serum or otherbiological samples from a subject. For purposes of the presentinvention, annexin proteins are characterized by a canonical motif inwhich a stretch of approximately 70 amino acids is repeated at leastfour times (Wallner, B. P. et al., 1986, Nature 320:77-80; Weber, K. andJohnson, N., 1986, FEBS Lett. 203: 95-98; Saris, C. J. M. et al., 1986,Cell 46:201-212; Huang K. S. et al., 1986, Cell 46:191-199).

Specifically, the invention encompasses a method for diagnosis andprognosis of cancer in a subject comprising:

(a) quantitatively detecting annexin protein in a biological samplederived from a subject; and

(b) comparing the level of protein detected in the subject's sample tothe level of protein detected in a control sample,

wherein an increase in the level of annexin protein detected in thesubject's sample as compared to control samples is an indicator of asubject with cancer or at increased risk for cancer. In addition todetecting annexin protein, annexin derived peptides, antigens ordifferentially modified annexin proteins may be detected for diagnosisand/or prognosis of cancer.

A wide variety of protein mixtures that may contain annexin proteins canbe prepared or assayed for the level of protein expression. In apreferred embodiment tumor tissue samples, including but not limited tolung tissue samples, derived from subjects suspected of, or predisposedto cancer can be used to screen for increased levels of annexin proteinproduction.

The present invention encompasses a method for diagnosis and prognosisof a subject with cancer, comprising:

(a) contacting an antibody-containing biological sample derived from asubject, with a sample containing annexin protein antigens underconditions such that an immunospecific antigen-antibody binding reactioncan occur; and

(b) detecting the presence of immunospecific binding of autoantibodiespresent in the subject's biological sample to the annexin protein,

wherein the presence of immunospecific binding of autoantibodiesindicates the presence of cancer in the subject.

In a specific embodiment of invention, annexin proteins, including butnot limited to Annexin I and II and peptides derived therefrom, arepurified and utilized to screen a subject's serum for the presence ofcirculating autoantibodies to such protein antigens, by means ofsensitive and rapid immunoadsorbent assays or by other procedures.

The present invention also provides for kits for carrying out theabove-described methods. The methods can be performed, for example, byutilizing pre-packaged diagnostic kits comprising at least a reagent fordetecting annexin protein such as an anti-annexin antibody.Alternatively, the diagnostic kits comprise an annexin protein orantigenic protein fragment for detection of annexin autoantibodies in asubject derived sample.

The present invention is based on the discovery that levels of Annexin Iand II proteins are increased in adenocarcinoma and squamous cell lungtumors derived from subjects with cancer. Additionally, increased levelsof circulating autoantibodies reactive against annexin proteins havebeen detected in the serum of subjects having adenocarcinoma andsquamous cell lung tumors.

5.1. Assays for Detection of Annexin Production

In accordance with the invention, measurement of levels of annexinproteins in samples derived from a subject can be used for the earlydiagnosis of diseases such as cancer. Moreover, the monitoring andquantitation of annexin protein levels can be used prognostically tostage the progression of the disease and to evaluate the efficacy ofagents used to treat a cancer subject.

The detection of annexin proteins in a sample from a subject can beaccomplished by any of a number of methods. Preferred diagnostic methodsfor the detection of annexin proteins in the biological sample of asubject can involve, for example, immunoassays wherein annexin proteinsare detected by their interaction with an annexin specific antibody.Antibodies useful in the present invention can be used to quantitativelyor qualitatively detect the presence of annexins or antigenic fragmentsthereof. In addition, reagents other than antibodies, such as, forexample, polypeptides that bind specifically to annexin proteins can beused in assays to detect the level of annexin protein expression.Alternatively, detection of annexin proteins may be accomplished bydetection and measurement of levels of biological properties associatedwith annexin proteins, such as for example, phopholipase A, andanticoagulant activity.

Immunoassays useful in the practice of the invention include but are notlimited to assay systems using techniques such as Western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, precipitin reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement fixation assays, immunoradiometric assays,fluorescent immunoassays, protein A immunoassays, to name but a few.

A biological sample which may contain annexin proteins, such as lungtissue or other biological tissue, is obtained from a subject suspectedof having a particular cancer or risk for cancer. Aliquots of wholetissues, or cells, are solubilized using any one of a variety ofsolubilization cocktails known to those skilled in the art. For example,tissue can be solubilized by addition of lysis buffer comprising (perliter) 8 M urea, 20 ml of Nonidet P-40 surfactant, 20 ml of ampholytes(pH 3.5-10), 20 ml of 2-mecaptoethanol, and 0.2 mM ofphenylmethylsulfonyl fluoride (PMSF) in distilled deionized water.

Immunoassays for detecting expression of annexin protein typicallycomprise contacting the biological sample, such as a tissue samplederived from a subject, with an anti-annexin antibody under conditionssuch that an immunospecific antigen-antibody binding reaction can occur,and detecting or measuring the amount of any immunospecific binding bythe antibody. In a specific aspect, such binding of antibody, forexample, can be used to detect the presence and increased production ofannexin proteins wherein the detection of increased production ofannexin proteins is an indication of a diseased condition. The levels ofannexin protein in a biological sample are compared to norms establishedfor age and gender-matched normal individuals and for subjects with avariety of non-cancerous or pre-cancerous disease states.

In an embodiment of the invention, the biological sample, such as atissue extract is brought in contact with a solid phase support orcarrier, such as nitrocellulose, for the purpose of immobilizing anyproteins present in the sample. The support is then washed with suitablebuffers followed by treatment with detectably labeled annexin specificantibody. The solid phase support is then washed with the buffer asecond time to remove unbound antibody. The amount of bound antibody onthe solid support is then determined according to well known methods.Those skilled in the art will be able to determine optional assayconditions for each determination by employing routine experimentation.

One of the ways in which annexin antibodies can be detectably labeled isby linking the antibody to an enzyme, such as for use in an enzymeimmunoassay (EIA) (Voller, A., “The Enzyme Linked Immunosorbent Assay(ELISA)”, 1978, Diagnostic Horizons 2:1-7, Microbiological AssociatesQuarterly Publication, Walkersville, Md.; Voller, A., et al., 1978, J.Clin. Pathol. 31:507-520; Butler, J. E., 1981, Meth. Enzymol.73:482-523). The enzyme which is bound to the antibody will react withan appropriate substrate, preferably a chromogenic substrate, in such amanner as to produce a chemical moiety that can be detected, forexample, by spectrophotometric, fluorimetric, or by visual means.Enzymes that can be used to detectable label the antibody include, butare not limited to, horseradish peroxidase and alkaline phosphatase.Detection can also be accomplished by colorimetric methods that employ achromogenic substrate for the enzyme.

Detection of annexin antibodies may also be accomplished using a varietyof other methods. For example, by radioactively labeling the antibodiesor antibody fragments, it is possible to detect annexin proteinexpression through the use of a radioimmunoassay (RIA) (see, forexample, Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society,March 1986). The radioactive isotope can be detected by such means asthe use of a gamma counter or a scintillation counter or byautoradiography.

The antibody may also be labeled with a fluorescent compound. Among themost commonly used fluorescent labeling compounds are fluoresceinisothiocyanate, rhodamine, phycoerythrin and fluorescamine. Likewise, abioluminescent compound may be used to label the annexin antibody. Thepresence of a bioluminescence protein is determined by detecting thepresence of luminescence. Important bioluminescence compounds forpurposes of labeling are luciferin, luciferase and aequorin.

In a specific embodiment of the invention, the levels of annexinproteins in biological samples can be analyzed by two-dimensional gelelectrophoresis. Methods of two-dimensional electrophoresis are known tothose skilled in the art. Biological samples, such as tissue samples,are loaded onto electrophoretic gels for isoelectric focusing separationin the first dimension which separates proteins based on charge. Anumber of first-dimension gel preparations may be utilized includingtube gels for carrier ampholytes-based separations or gels strips forimmobilized gradients based separations. After first-dimensionseparation, proteins are transferred onto the second dimension gel,following an equilibration procedure and separated using SDS PAGE whichseparates the proteins based on molecular weight. When comparingbiological samples derived from different subjects, multiple gels areprepared from individual biological samples (including samples fromnormal controls).

Following separation, the proteins are transferred from thetwo-dimensional gels onto membranes commonly used for Western blotting.The techniques of Western blotting and subsequent visualization ofproteins are also well known in the art (Sambrook et al, “MolecularCloning, A Laboratory Manual”, 2^(nd) Edition, Volume 3, 1989, ColdSpring Harbor). The standard procedures may be used, or the proceduresmay be modified as known in the art for identification of proteins ofparticular types, such as highly basic or acidic, or lipid soluble, etc.(See for example, Ausubel, et al., 1999, Current Protocols in MolecularBiology, Wiley & Sons, Inc., N.Y.). Antibodies that bind to the annexinproteins are utilized in an incubation step, as in the procedure ofWestern blot analysis. A second antibody specific for the first antibodyis utilized in the procedure of Western blot analysis to visualizeproteins that reacted with the first antibody.

The detection of annexin protein levels in biological samples can alsobe used to monitor the efficacy of potential anti-cancer agents duringtreatment. For example, the level of annexin protein production can bedetermined before and during treatment. The efficacy of the agent can befollowed by comparing annexin expression throughout the treatment.Agents exhibiting efficacy are those which decrease the level of annexinprotein production as treatment with the agent progresses.

The present invention is demonstrated by way of example wherein elevatedlevels of annexin proteins have been detected in tissue samples derivedfrom patients with lung adenocarcinoma and squamous cell lung carcinoma.In particular, increased levels of Annexin I and II were detected insamples derived from lung cancer patients. The detection and/orquantitative measurement of annexin proteins in biological samples canbe used in screening of subjects who are at risk for developing certaintypes of cancers or other proliferative disorders in which the annexinproteins are overexpressed. In addition, qualitative differences in thepattern of occurrence in serum or biological fluids of different membersof the annexin family of proteins can be used as a screening, diagnosticor prognostic indicator of cancer or cancer risk.

5.2. Assays for Detection of Anti-Annexin Autoantibodies

The present invention provides diagnostic and prognostic methods fordiseases such as cancer based on detection of circulating annexinautoantibodies in a subject. The method is validated by the use of abiological sample from a subject with cancer and from age and gendermatched controls, without cancer. A biological sample which may containautoantibodies, such as serum, is obtained from a subject suspected ofhaving a particular cancer or suspected of being predisposed todeveloping cancer. A similar body fluid is obtained from a controlsubject that does not have cancer.

In accordance with the invention, measurement of autoantibodies reactiveagainst the annexin protein antigens can be used for the early diagnosisof diseases such as cancer. Moreover, the monitoring of autoantibodylevels can be used prognostically to stage the progression of thedisease. The detection of autoantibodies in a serum sample from apatient can be accomplished by any of a number of methods. Such methodsinclude immunoassays which include, but are not limited to, assaysystems using techniques such as Western blots, radioimmunoassays, ELISA(enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few.

Such an immunoassay is carried out by a method comprising contacting aserum sample derived from a subject with a sample containing the annexinprotein antigens under conditions such that an immunospecificantigen-antibody binding reaction can occur, and detecting or measuringthe amount of any immunospecific binding by the autoantibody. In aspecific aspect, such binding of autoantibody by tissue sections, forexample, can be used to detect the presence of autoantibody wherein thedetection of autoantibody is an indication of a diseased condition. Thelevels of autoantibodies in a serum sample are compared to the levelspresent in an analogous serum sample from a subject not having thedisorder.

The immunoassays can be conducted in a variety of ways. For example, onemethod to conduct such assays involves anchoring of annexin protein ontoa solid support and detecting anti-annexin antibodies specifically boundthereto. The annexin proteins to be utilized in the assays of theinvention can be prepared via recombinant DNA techniques well known inthe art. For example, a DNA molecule encoding an annexin protein or anantigenic fragment thereof can be genetically engineered into anappropriate expression vector for large scale preparation of annexinprotein. It may be advantageous to engineer fusion proteins that canfacilitate labeling, immobilization or detection of the annexin protein.See, for example, the techniques described in Sambrook et al., 1989,Molecular Cloning: A laboratory Manual, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. Alternatively, the annexin protein may be purifiedfrom natural sources, e.g., purified from cells, using proteinseparation techniques well known in the art. Such purificationtechniques may include, but are not limited to molecular sievechromatography and/or ion exchange chromatography. In practice,microtitre plates are conveniently utilized as the solid support for theannexin proteins. The surfaces may be prepared in advance and stored.

The present invention is demonstrated by way of example wherein elevatedlevels of circulating autoantibodies reactive against several annexinprotein antigens have been detected in the sera of cancer patients. Thedetection and/or quantitative measurement of circulating anti-annexinautoantibodies in serum can be used in screening of subjects who are atrisk for cancer or other proliferative disorders in which annexinprotein levels are increased.

5.3. Immunotherapy

The invention also relates to the use of annexin proteins as antigens toimmunize patients suffering from diseases characterized by theproduction of the annexin protein antigens. Stimulation of animmunological response to such antigens, is intended to elicit a moreeffective attack on tumor cells; such as inter alia inhibiting tumorcell growth or facilitating the killing of tumor cells. Theidentification of autoantibodies to annexin protein antigens associatedwith particular cancers provides a basis for immunotherapy of thedisease.

The patient may be immunized with the annexin protein antigens to elicitan immune response which facilitates killing of tumor cells orinhibiting tumor cell growth. The annexin protein antigens can beprepared using the methods described above for purification of proteins.

In an embodiment of the invention an immunogen comprising a purifiedannexin protein antigen to which a patient with cancer has developedautoantibodies, is used to elicit an immune response. Foradministration, the annexin protein antigen should be formulated with asuitable adjuvant in order to enhance the immunological response to theprotein antigen. Suitable adjuvants include, but are not limited tomineral gels, e.g. aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, andpotentially useful human adjuvants such as BCG (bacilli Calmett-Guerin)and (Corynebacterium parvum). Many methods may be used to introduce theformulations derived above; including but not limited to oral,intradermal, intramuscular, intraperitoneal, intravenous, andsubcutaneous.

5.4. Kits

The present invention further provides for kits for carrying out theabove-described assays. The assays described herein can be performed,for example, by utilizing pre-packaged diagnostic kits, comprising atleast an annexin peptide (for detection of Annexin autoantibodies) or anannexin antibody reagent (for detection of annexin protein), which canbe conveniently used, e.g., in clinical settings to diagnose disorderssuch as cancer.

In a first series of nonlimiting embodiments, a kit according to theinvention comprises components for detecting and/or measuring human IgGantibodies directed toward annexin antigen. As one example, where theantibodies are detected and/or measured by enzyme linked immunoabsorbentassay (ELISA), such components may comprise target antigen, in the formof at least one and preferably a plurality of different annexin antigensor epitopes thereof, linked to a solid phase, and a means for detectinga human antibody bound to target antigen. Such means for detection maybe, for example, an antibody directed toward the constant region ofhuman IgG (e.g., rabbit anti-human IgG antibody), which may itself bedetectably labeled (e.g., with a radioactive, fluorescent, colorimetricor enzyme label), or which may be detected by a labeled secondaryantibody (e.g., goat anti-rabbit antibody).

In a second series of nonlimiting embodiments, a kit according to theinvention may comprise components which detect and/or measure annexinantigens in the biological sample of a subject. For example, whereannexin proteins are detected and/or measured by enzyme linkedimmunoabsorbent assay (ELISA), such components may comprise an antibodydirected to epitopes of the annexin proteins which can be used to detectand/or quantitate the level of annexin expression in the biologicalsample. The antibody itself may be detectably labeled with aradioactive, flourescent, colorimetric or enzyme label. Alternatively,the kit may contain a labeled secondary antibody.

6. EXAMPLE Detection of Autoantibodies Specific for Annexin Proteins andIncreased Levels of Annexin Protein Expression in Cancer Patient DerivedSamples

Using the methods of the present invention, sera obtained from subjectswith cancer were screened for reactivity against annexin proteins. Thesera samples from cancer subjects were found to be reactive againstannexin proteins. In addition, increased levels of annexin proteinproduction were observed in tissue samples derived from subjects withcancer.

6.1. Materials and Methods 6.1.1. Reagents

All cell culture reagents, including Dulbecco's modified Eagle's medium(DMEM, containing L-glutamine, sodium pyruvate and pyridoxinehydrochloride), Dulbecco's phosphate buffered saline (PBS), fetal calfserum and penicillin/streptomycin were obtained from GIBCO-BRL (GrandIsland, N.Y.). Mouse monoclonal anti-Annexin I and II antibodies wereobtained from ICN (Costa Mesa, Calif.). Mouse monoclonal anti-human IgMantibody was purchased from Sigma Chemical Company (St. Louis, Mo.).Horseradish peroxidase-conjugated mice anti-human IgG1, IgG2, IgG3 andIgG4 antibodies were purchased from Zymed Company (San Francisco,Calif.). Horseradish peroxidase-conjugated sheep anti-human IgG and theanti-mouse IgG monoclonal antibodies, the ECL (EnhancedChemiluminescence) kit and the Hyperfilm MP were obtained from Amersham(Arlington Heights, Ill.). Immobilon-P PVDF (polyvinylidene fluoride)membranes were purchased from Millipore Corp. (Bedford, Mass.). Theacrylamide used in the first dimension electrophoresis, urea, ammoniumpersulfate and PDA (piperazine diacrylamide) were all purchased fromBio-Rad (Rockville Center, N.Y.). The acrylamide used in the seconddimension electrophoresis was purchased from Serva (Crescent Chemical,Hauppauge, N.Y.) and the carrier ampholytes (pH 4 to 8) and NP-40 wereboth purchased from Gallard/Schlessinger (Carle Place, N.Y.). All otherreagents and chemicals were obtained from either Fisher or Sigma andwere of the highest purity available.

6.1.2. Cell Culture and Preparation of Extracts

A549 human adenocarcinoma cell lines were cultured at 37° C. in a 6%CO₂-humidified incubator in DMEM supplemented with 10% fetal calf serum,100 U/ml penicillin and 100 U/ml streptomycin. The cells were passagedweekly after they had reached 70-80% confluence.

Fresh tumor tissue was obtained at the time of diagnosis (i.e., biopsytissue) from patients with lung cancer. The experimental protocol wasapproved by the University of Michigan Institutional Review Board forApproved Research Involving Human Subjects. Informed consent wasobtained from the patients (or their families) prior to the study.Following excision, the tumor tissue was immediately frozen at −80° C.,after which small amounts of tumor tissue were solublized insolubilization buffer and stored at −80° C. until use. Cultured cellswere lysed by addition of 200 μl of solubilization buffer comprising of8 M urea, 2% NP-40, 2% carrier ampholytes (pH 4 to 8), 2%β-mecaptoethanol and 10 mM PMSF, and harvested using a cell scraper.After an additional 100 μl of solubilization buffer was added, thesolution containing the cell extracts was transferred into microfugetubes and stored at −80° C. until use.

6.1.3. 2-D Page and Western Blotting

Proteins derived from the extracts of both cultured cells or solidtumors were separated in dimensions as previously described (Strahler etal., 1989, Two-dimensional polyacrylamide gel electrophoresis ofproteins. In “Protein Structure: A Practical Approach” T. E. Creighton(ed.) IRL Press, Oxford, U.K., pp65-92), with some modifications.Briefly, subsequent to cellular lysis in solubilization buffer, 35 μlaliquots of solubilized tumor tissue or cultured cells derived fromapproximately 2.5×10⁶ cells were applied onto isofocusing gels.Isoelectric focusing was conducted using pH 4 to 8 carrier ampholytes at700 V for 16 h, followed by 1000 V for an additional 2 h. Thefirst-dimension tube gel was loaded onto a cassette containing thesecond dimension gel, after equilibration in second-dimension samplebuffer (125 mM Tris (pH 6.8), containing 10% glycerol, 2% SDS, 1%dithiothreitol and bromophenol blue). For the second-dimensionseparation, an acrylamide gradient of 11% to 14% was used, and thesamples were electrophoresed until the dye front reached the oppositeend of the gel. The separated proteins were transferred to anImmobilon-P PVDF membrane. Protein patterns in some gels were visualizedby silver staining, and on some Immobilon-P membranes by coomassieblue-staining of the membranes. Unstained membranes prepared forhybridization were incubated with blocking buffer (comprising Trisbuffered saline (TBS) containing 1.8% nonfat dry milk and 0.1% Tween 20)for 2 h, then washed and incubated with serum obtained either frompatients, or with normal control serum (300 μl of serum, at a 1:100dilution) for 1 h at room temp. Following three washes with blockingbuffer, the membranes were incubated with a horseradishperoxidase-conjugated sheep anti-human IgG antibody (at a 1:1000dilution) for 30 min at room temp. The membranes were washed 5 timeswith TBS containing 0.1% Tween 20, once in TBS, briefly incubated in ECLand exposed to hyperfilm™ MP for 10-30 min. Patterns visualized afterhybridization with patients sera were compared directly to both thecoomassie blue-stained blots from the same sample to determinecorrelation with proteins, as well as to patterns obtained fromhybridization of blots derived from the same sample with sera frompatients with other solid tumors or control sera to determine thespecificity of autoantigens. Alternatively membranes were incubated witha horseradish peroxidase-conjugated sheep anti-human IgM antibody andprocessed as for incubations with anti-human IgG antibody.

Protein spots in both tumor and lysates of lung adenocarcinoma celllines that were visualized with patient sera, but not with control sera,were characterized further. A549 cell lysates were subjected to 2-DPAGE, after which the separated proteins were transferred to Immobilon-Pmembranes and subsequently stained with coomassie blue. Protein spots ofinterest were excised from the membrane and subjected to N-terminalamino acid sequencing and mass spectrometric analysis. The resultantsequences and peptide masses were utilized for database searches forprotein identification.

6.1.4. Annexin I and II Detection by Immunoblotting

Two anti-Annexin I and II monoclonal antibodies were utilized. Theseprimary antibodies were used at a 1:5000 dilution in immunoblottingassays and processed as for incubations with patient sera.

6.2. Results 6.2.1. Reactivity of Sera from Lung Cancer Patients with aLung Cancer Protein Detected by Western Blot Analysis

A549 cell proteins were separated by 2-D PAGE and transferred ontoImmobilon-P PVDF membranes. For Western blot analysis, each membrane wastreated with one serum sample. The samples included sera obtained at thetime of diagnosis from 18 patients with lung adenocarcinoma, 11 patientswith squamous cell lung carcinoma, 4 with small cell lung carcinoma, 2with large cell lung carcinoma, 19 with unclassified lung cancer; 37patients with other cancers (11 breast, 7 melanoma, 11 liver, and 8esophageal cancer; and from 15 healthy subjects without a prior historyof cancer or autoimmune disease.

An example of a 2-D gel of A549 cells stained with silver is shown inFIG. 1. Hybridization of membranes using patient sera as the primaryantibody and sheep anti-human IgG as secondary antibody revealedvariable patterns of reactivity among patient sera with lung cancer.Duplicate hybridizations resulted in similar patterns. In general,several reactive spots were observed with most sera. Some of thereactive spots were observed with control sera and thus were consideredto represent non-specific reactivity. Others were restricted to patientsera with lung cancer. Most noticeable among the latter was intensereactivity in a group of contiguous protein spots designated A1, with apI between 6.3 and 6.8 M.W. of 37 kDa (FIG. 2), which was observed withsera of 8 of 18 lung adenocarcinoma patients, 4 of 11 squamous cell lungcarcinoma patients, 1 of 4 small cell lung carcinoma, and 2 of 19unclassified lung cancer patients and which was absent in A549 membraneshybridized with sera from normal individuals. A second group ofcontiguous protein spots designated A2 (FIG. 3), with a pI between 7.2and 7.8 and MW of approximately 36 kDa was observed with sera of 7 of 18lung adenocarcinoma patients, 3 of 11 squamous cell lung carcinomapatients, 2 of 4 small cell lung carcinoma patients, and 6 of 19unclassified lung cancer patients but absent in sera from normalindividuals. In total, 11 of 18 sera from patients with lungadenocarcinoma and 6 of 11 sera from patients with squamous cell lungcarcinoma and 3 of 4 with small cell lung carcinoma exhibited reactivityfor A1 and/or A2.

6.2.2. IgM Immunoreactivity Against Lung Cancer Proteins

To determine whether sera that exhibited IgG based immunoreactivityagainst A1 and/or A2 proteins also exhibited IgM based immunoreactivity,serum from three patients with lung adenocarcinoma that exhibited IgGbased immunoreactivity against A1 and/or A2 and from two negativecontrols were included in this analysis. Membranes containing A549lysates were hybridized with patient and control sera and subsequentlyincubated with a horseradish peroxidase-conjugated sheep anti-human IgMantibody. Only the patient serum that exhibited IgG based reactivityagainst A1 and A2 exhibited IgM based reactivity against these twocontiguous set proteins (FIG. 3).

6.2.3. IgG Subtypes Immunoreactivity Against Lung Cancer Proteins

To determine sera IgG subtype that exhibited IgG based immunoreactivityagainst A1 and/or A2 proteins, serum from three patients with lungadenocarcinoma that exhibited IgG immunoreactivity against A1 and/or A2proteins and from two negative controls were included in this analysis.Membranes containing A549 lysates were hybridized with patient andcontrol sera and subsequently incubated with a horseradishperoxidase-conjugated mouse anti-human IgG1, IgG2, IgG3 or IgG4antibody. Only the patient sera that exhibited IgG based reactivityagainst A1 and/or A2 exhibited IgG1 based reactivity against these twocontiguous sets of proteins (FIG. 4).

6.2.4. Specificity of A1 and A2 IgG Antibodies to Lung Cancer

Membranes containing A549 lysates were hybridized with serum frompatients with melanoma, breast cancer, liver cancer or esophagealcancer. None of the serum exhibited IgG based immunoactivity against A2.However, the serum from patients with esophageal cancer (5/8) and breastcancer (1/11) showed immunoactivity against the Al proteins.

6.2.5. Reactivity or Lung Cancer Patient Sera with Annexin I and IIIsoforms

To determine the identity of the A1 and A2 proteins, additionalmembranes were prepared from A549 lung adenocarcinoma cell lysates andthe proteins were visualized by Coomasie blue staining. The A1 and A2spots were excised from the membranes, eluted and subjected to trypsindigestion. Peptide fragments were analyzed by Mass Spectrometry. Fourcontiguous sections, designated A1a, A1b (just to the left of A1a, i.e.more acidic), A1c (just to the left of A1b, i.e. more acidic) and A1d(just to the left of A1c, i.e. more acidic) were excised from the blots.These four areas comprised, in total, the entire A1 region which wasimmunoreactive. Mass Spectrometric analysis of peptides derived fromthese sections revealed that Annexin I and Annexin II variants were thepredominant Annexin I isoforms present except section A1a which producedno result. For A2 proteins, two contiguous sections, designated A2a andA2b (just to the left of A1b, i.e. more acidic) were excised from theblots. Mass Spectrometric analysis of peptides derived from thesesections revealed that Annexin II variant (A2a) and Annexin II variant(A2b) were the predominant Annexin II isoforms present Additionally, A1and A2 spots excised from the membranes and subjected to directN-terminal sequencing. Since annexins are N-terminally blocked byacetylation, no results were obtained.

6.2.6. Further Confirmation of the Annexin I and II Isoforms

To confirm that spots A1 and A2 consisted of a mixture of Annexin I andAnnexin II respectively, membranes prepared from both the A549adenocarcinoma cell line were hybridized with either a commerciallyavailable mouse monoclonal antibody that reacts with Annexin I, or themouse monoclonal Annexin II antibody. The immunoreactivity patternsobtained were compared to both coomassie blue-stained blots of the samecell lysate type as well as to the patterns of immunoreactivity observedwith sera from patients with lung cancer. Anti-Annexin I, andanti-Annexin II antibodies immunoreacted with proteins in the A1 and A2spots respectively visualized with lung cancer patient sera (FIG. 6).Strikingly, several additional low molecular weight immunoreactive spotswere observed resulting from the hybridization of both monoclonalanti-Annexin I and II antibodies utilized. These spots have not shownimmunoreactivity with any patient sera tested.

The present invention is not to be limited in scope by the embodimentsdisclosed in the examples which are intended as an illustration of oneaspect of the invention, and any compositions or methods which arefunctionally equivalent are within the scope of this invention. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description. Such modifications are intended to fallwithin the scope of the claims.

We claim:
 1. A method for diagnosing lung cancer in a subjectcomprising: (i) contacting a serum sample derived from the subject withone or more protein antigens wherein said protein antigens are Annexin Iand Annexin II protein antigens; and (ii) detecting immunospecificbinding of Annexin I or Annexin II autoantibodies in the serum sample,wherein an increase in the level of immunospecific binding detected instep (ii) as compared to a control serum sample is an indicator of lungcancer.
 2. A method for diagnosing breast cancer in a subjectcomprising: (i) contacting a serum sample derived from the subject withone or more protein antigens wherein said protein antigens are Annexin Iantigens; and (ii) detecting immunospecific binding of Annexin Iautoantibodies in the serum sample, wherein an increase in the level ofimmunospecific binding detected in step (ii) as compared to a controlserum sample is an indicator of breast cancer.
 3. A method fordiagnosing esophageal cancer in a subject comprising: (i) contacting aserum sample derived from the subject with one or more protein antigenswherein said protein antigens are Annexin I antigens; and (ii) detectingimmunospecific binding of Annexin I autoantibodies in the serum sample,wherein an increase in the level of immunospecific binding detected instep (ii) as compared to a control serum sample is an indicator ofesophageal cancer.
 4. The method of claim 1, 2 or 3 wherein the AnnexinI or Annexin II autoantibodies are detected using an immunoassay.
 5. Themethod of claim 4 wherein the Annexin I and Annexin II autoantibodiesare detected using an immunoassay comprising: (a) immobilizing one ormore annexin proteins on a substrate or membrane wherein said annexinproteins are Annexin I and Annexin II; (b) contacting the substrate ormembrane with the subject's serum sample; and (c) detecting the presenceof Annexin I and Annexin II autoantibodies bound to the substrate. 6.The method of claim 4 wherein the presence of Annexin I and Annexin IIautoantibodies is detected using a labeled reagent that binds to theautoantibodies.